The effect of different conditions on protoplast formation was studied in the streptomycin-resistant strain Cellulomonas sp. M32Bo. The greatest efficiency (75% protoplasts) was achieved by use of 0.5 M sodium succinate as osmotic stabilizer, supplemented with 20 mM MgCl2, 200-mu-g/ml of lysozyme, and 0.01 M EDTA at pH 7.4. Cells harvested at the mid-exponential growth phase were more suitable for protoplast formation than those of the stationary phase. Electron microscopy observations showed the presence of both protoplasts and spheroplasts in the treated samples, some of them still showing a rod shape. Two regeneration media were developed that showed similar regeneration frequencies (52%). Strain M32Bo was fused with a tetracycline-resistant strain (Cellulomonas sp. Sz). Segregation analysis of fusant colonies suggested the existence of a temporary diploid stage in which both parental genotypes were expressed.